SNPSIG SARS-COV-2 (20H/501Y.V2) - SOUTH AFRICA VARIANT - GENESIG
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Primerdesign Ltd
SNPsig® SARS-CoV-2
(20H/501Y.V2)
South Africa variant
SNPsig® real-time PCR
SARS-CoV-2 mutation detection/
allelic discrimination kit
96 tests
For general laboratory and research use only
Detection of 20H/501Y.V2 (South Africa variant) 1
SNPsig kit handbook Path-SNPsig-SARS-CoV-2-20H/501Y.V2—STED version 1.03
Published Date: 24/03/2021
Kit contents
• SARS-CoV-2 (20H/501Y.V2) genotyping primer/probe mix (96 reactions BROWN)
FAM and HEX labelled
• Wild-type positive control template (RED)
• Mutant positive control template (RED)
• RNase/DNAse free water (WHITE)
for resuspension of primer/probe mix
• Template preparation buffer (YELLOW)
for resuspension of positive control templates
• Onestep lyophilised mastermix (RED)
contains complete Onestep RT-qPCR mastermix
• Mastermix resuspension buffer (BLUE)
for resuspension of the lyophilized mastermix
Reagents and equipment to be supplied
by the user
Real-time PCR Instrument
Must be able to read fluorescence through FAM and HEX channels (plus Cy5 if using optional
internal control)
Pipettes and tips
Vortex
Centrifuge
Suitable qPCR 96W plates or qPCR reaction tubes
Detection of 20H/501Y.V2 (South Africa variant) 2
SNPsig kit handbook Path-SNPsig-SARS-CoV-2-20H/501Y.V2—STED version 1.03
Published Date: 24/03/2021Kit storage and stability
This kit is stable at room temperature but should be stored at -20ºC on arrival. Primerdesign
does not recommend using the kit after the expiry date stated on the pack. Once the
lyophilised components have been resuspended, unnecessary repeated freeze/thawing
should be avoided. The kit is stable for six months from the date of resuspension under these
circumstances.
Suitable sample material
SNPsig SARS-CoV-2 SA SNP is intended for use as a reflex test only. Thus, a primary
confirmation test for SARS-CoV-2 would be carried out using suitable methodology, and the
extracted RNA from patient samples (or any material suited for PCR amplification) thereafter
applied to this test. Please ensure that the samples are suitable in terms of purity,
concentration, and RNA integrity. Always run at least one negative control with the samples.
To prepare a negative control, replace the template RNA sample with RNase/DNAse free
water.
Notices and disclaimers
This product is developed, designed and sold for research purposes only. It is not intended
for human diagnostic or drug purposes or to be administered to humans unless clearly
expressed for that purpose by the Food and Drug Administration in the USA or the
appropriate regulatory authorities in the country of use. During the warranty period
Primerdesign SNPsig detection kits allow precise and reproducible data recovery combined
with excellent sensitivity. For data obtained by violation to the general GLP guidelines and the
manufacturer’s recommendations the right to claim under guarantee is expired. PCR is a
proprietary technology covered by several US and foreign patents. These patents are owned
by Roche Molecular Systems Inc. and have been sub-licensed by PE Corporation in certain
fields. Depending on your specific application you may need a license from Roche or PE to
practice PCR. Additional information on purchasing licenses to practice the PCR process may
be obtained by contacting the Director of Licensing at Roche Molecular Systems, 1145
Atlantic Avenue, Alameda, CA 94501 or Applied Biosystems business group of the Applera
Corporation, 850 Lincoln Centre Drive, Foster City, CA 94404. In addition, the 5' nuclease
assay and other homogeneous amplification methods used in connection with the PCR
process may be covered by U.S. Patents 5,210,015 and 5,487,972, owned by Roche
Molecular Systems, Inc, and by U.S. Patent 5,538,848, owned by The Perkin-Elmer
Corporation.
Trademarks
PrimerdesignTM is a trademark of Primerdesign Ltd.
The PCR process is covered by US Patents 4,683,195, and 4,683,202 and foreign
equivalents owned by Hoffmann-La Roche AG. BI, ABI PRISM® GeneAmp® and MicroAmp®
are registered trademarks of the Applera Genomics (Applied Biosystems Corporation).
BIOMEK® is a registered trademark of Beckman Instruments, Inc.; iCycler™ is a registered
trademark of Bio- Rad Laboratories, Rotor-Gene is a trademark of Corbett Research.
LightCycler™ is a registered trademark of the Idaho Technology Inc. GeneAmp®, TaqMan®
and AmpliTaqGold® are registered trademarks of Roche Molecular Systems, Inc., The
purchase of the Primerdesign reagents cannot be construed as an authorization or implicit
license to practice PCR under any patents held by Hoffmann-LaRoche Inc.
Detection of 20H/501Y.V2 (South Africa variant) 3
SNPsig kit handbook Path-SNPsig-SARS-CoV-2-20H/501Y.V2—STED version 1.03
Published Date: 24/03/2021Introduction
The Novel Coronavirus Disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2 virus,
represents a major threat to health. COVID-19 has resulted in widespread morbidity and
mortality. SARS-CoV-2 is known to have infected more than 90 million people (1). As the
virus has spread across the world, mutations have arisen resulting in divergent clusters
(clades) with different prevalence in different geographic regions (2,3).
In South Africa, one lineage has spread rapidly, quickly becoming the dominant strain,
known as 20H/501Y.V2 or B.1.351 (4). This variant is characterized by eight lineage-
defining mutations in the spike protein, including mutations at key residues (E484K,
N501Y) which have been implicated in increased transmissibility and antibody evasion (5–
7). In addition, the variant E484K raised concerns over the potential to impact vaccine
efficacy (8–10). Rates of reinfection and potential for increased disease severity
suggested for this variant have raised concern with public health bodies internationally.
As of 24th January 2021, 20H/501Y.V2 had been reported in 31 countries (11). Prevention
of community outbreaks will need to be prioritised to ensure this lineage does not become
more prevalent and a greater burden of COVID-19 in the health sector. Novacyt performed
a thorough bioinformatic investigation to find unique identifiers to 20H/501Y.V2 variant and
selected the optimal Single-Nucleotide Polymorphism (SNP) to identify this variant (SA
SNP). Targeting of SA SNP is 100% specific for this strain and will allow targeted testing
of positive SARS-CoV-2 samples to find the 20H/501Y.V2 variant.
Detection of 20H/501Y.V2 (South Africa variant) 4
SNPsig kit handbook Path-SNPsig-SARS-CoV-2-20H/501Y.V2—STED version 1.03
Published Date: 24/03/2021Principles of the test
Genotyping by real-time PCR using hydrolysis probes
Each genotyping primer/probe mix contains two labelled probes homologous to the two
genotypes under investigation. During qPCR amplification of the reverse-transcribed target
RNA, the probes will compete for binding across the variant region. The probe that is 100%
homologous to the binding site will preferentially bind and give a fluorescent signal as PCR
proceeds. It follows that the wild-type sequence will give a strong amplification plot through
one channel whilst giving a very weak signal through the alternative channel. Variant
samples will give an exactly inverse result. Most hardware platforms can perform this
analysis automatically. The SNPsig® assays are compatible with all qPCR instruments
capable of detecting fluorescence in FAM and HEX (plus Cy5 if using internal control)
emission channels, including selected genesig® family instruments.
Positive controls
The kit contains positive control templates for each of the two genotypes. These can be run as
parallel samples to give control signals for each genotype. Positive control templates are a
potential contamination risk to subsequent tests so must be handledcarefully.
Negative control
To confirm the absence of contamination, a negative control reaction should be included
every time the kit is used. In this instance, the RNase/DNAse free water should be used
instead of template RNA. A negative result in FAM and HEX channels indicates that the
reagents have not become contaminated while setting up the run. If a positive result is
obtained, this indicates contamination, therefore the sample results will be invalid and the
run should be repeated. Possible sources of contamination should first be explored and
removed.
Master mix compatibility
Onestep lyophilised mastermix contains the enzyme, nucleotides, buffers and salts at
precisely the correct concentration for this application. The annealing temperatures of the
primer and probe have been carefully calibrated and any change in the reaction buffer can
significantly alter the performance of the assay. For this reason, Primerdesign can only
guarantee accurate genotyping results when Onestep lyophilised mastermix is used.
Detection of 20H/501Y.V2 (South Africa variant) 5
SNPsig kit handbook Path-SNPsig-SARS-CoV-2-20H/501Y.V2—STED version 1.03
Published Date: 24/03/2021Resuspension Protocol
To minimize the risk of contamination with foreign RNA, we recommend that all pipetting be
performed in a PCR clean environment. Ideally this would be a designated PCR lab or PCR
cabinet. Filter tips are recommended for all pipetting steps. The positive control template is a
significant contamination risk and should therefore be pipetted after negative control and
sample wells.
1. Pulse-spin each tube in a centrifuge before opening.
This will ensure lyophilised primer and probe mix is in the base of the tube and is not
lost upon opening the tube.
2. Resuspend the primer/probe mix in the RNase/DNAse free water supplied,
according to the table below.
To ensure complete resuspension, vortex the tube thoroughly.
Component - Resuspend in water Volume
SARS-CoV-2 (20H/501Y.V2) primer/probe mix (BROWN) 110 µl
3. Resuspend the mastermix in resuspension buffer supplied, according to the
table below.
Component - Resuspend in resuspension buffer Volume
Onestep lyophilised mastermix (RED) 525 µl
4. Resuspend the positive control templates in the template preparation buffer
supplied, according to the table below.
To ensure complete resuspension, vortex each tube thoroughly.
Component - Resuspend in template preparation buffer Volume
Wild-type Positive Control Template (RED) * 500 µl
Mutant Positive Control Template (RED) * 500 µl
* This component contains high copy number template and is a VERY significant
contamination risk. It must be opened and handled in a separate laboratory environment,
away from the other components.
Detection of 20H/501Y.V2 (South Africa variant) 6
SNPsig kit handbook Path-SNPsig-SARS-CoV-2-20H/501Y.V2—STED version 1.03
Published Date: 24/03/2021qPCR detection protocol
If using optional internal control, refer to page 11 for protocol amendments.
1. Prepare a complete genotyping reaction mix for the primer/probe mix
according to the table below:
Include sufficient reactions for all samples, positive and negative controls.
Component Volume
Onestep lyophilised mastermix 10 µl
SARS-CoV-2 (20H/501Y.V2) primer/probe mix (BROWN) 1 µl
RNase/DNAse free water (WHITE) 4 µl
Final Volume 15 µl
2. Pipette 15µl of this mix into each well according to your qPCR experimental plate
set up.
3. Prepare RNA templates for each of your samples.
As this is a reflex test, the RNA obtained from the confirmatory primary assay for
SARS-CoV-2 should be used.
4. Pipette 5µl of RNA template into each well, according to your experimental
plate set up.
For negative control wells use 5µl of RNase/DNAse free water. The final volume in
each well is 20µl.
5. Pipette 5µl of each positive control RNA according to your experimental plate
set up.
Detection of 20H/501Y.V2 (South Africa variant) 7
SNPsig kit handbook Path-SNPsig-SARS-CoV-2-20H/501Y.V2—STED version 1.03
Published Date: 24/03/2021qPCR amplification protocol
The following protocol is recommended for optimum resolution between genotypes:
Protocol for Onestep lyophilised mastermix
Step Time Temp
Reverse transcription 10 min 55 oC
Enzyme activation 2 min 95 oC
Cycling x45 Denaturation 10s 95 oC
Annealing and extension (DATA
60s 60 oC
COLLECTION) *
* Fluorogenic data should be collected during this step through the FAM and HEX channels
Detection of 20H/501Y.V2 (South Africa variant) 8
SNPsig kit handbook Path-SNPsig-SARS-CoV-2-20H/501Y.V2—STED version 1.03
Published Date: 24/03/2021Interpretation of results
The wild-type probe is labelled to read through the FAM channel whilst the mutant probe is
labelled to read through the HEX channel. On wild-type sequences the FAM channel will give
a strong amplification plot and the HEX channel none or very low detection. The signals are
reversed on mutant samples. Cq values >40 should be disregarded and counted as
inconclusive.
If using an internal control (see protocol below), then the Cy5 channel should be consulted
where the test is negative in both the FAM and HEX channels. If neither HEX or FAM amplify,
but Cy5 channel is observed, then the reaction worked, and the test is a true negative. If no
Cy5 amplification is observed, then the test failed.
Sample data
Wild Type sample (WT signal, Mutant signal)
Amplification in the FAM (Blue) channel with no amplification in the HEX (green) channel
indicates the presence of wild type template only.
Detection of 20H/501Y.V2 (South Africa variant) 9
SNPsig kit handbook Path-SNPsig-SARS-CoV-2-20H/501Y.V2—STED version 1.03
Published Date: 24/03/2021Variant RNA sample (WT signal, Mutant signal)
NTC HEX amplification shown in red
Amplification in the HEX (green) channel with no amplification in the FAM (blue) channel
indicates the presence of mutant template only.
If the HEX background fluorescence is high, viewed as a low-level increase in fluorescence
which is not sigmoid in shape (curve indicated in red), view the amplification plots in log scale
and raise the threshold manually in order to exclude it.
The raw data above can best be visualised by using a cluster analysis; plotting the end point
fluorescence data from the FAM channel on one axis and the end point fluorescence data
from the HEX channel on the other axis. Most qPCR software platforms will perform this
analysis automatically so follow the manufacturer’s instructions for your software. The data
are quickly resolved into clusters corresponding to the wild-type, and mutant variant samples.
Detection of 20H/501Y.V2 (South Africa variant) 10
SNPsig kit handbook Path-SNPsig-SARS-CoV-2-20H/501Y.V2—STED version 1.03
Published Date: 24/03/2021Optional: Use of Internal Control (CY5)
Internal RNA control (CY5) is available as a separate product, compatible with this kit
(Product Code: Z-INT-RNA-CY5-LL).
To confirm the occurrence of PCR, an internal control reaction can be used. Successful
real-time PCR amplification of the internal control indicates that PCR inhibitors are not
present at a high concentration. An internal control primer/probe mix labelled with the CY5
fluorophore can be used with this kit to detect the exogenous RNA template following
cDNA synthesis using qPCR. The primers and CY5 probe allow detection of the control
cDNA alongside the target cDNA in a multiplex reaction. Amplification of the control cDNA
does not interfere with detection of the target gene even when the target gene is present
at low copy number. If a positive result is obtained, this indicates that the reaction was
successful and that any negative result for the primary reaction is a true negative.
Resuspension Protocol
To minimize the risk of contamination with foreign RNA, we recommend that all pipetting be
performed in a PCR clean environment. Ideally this would be a designated PCR lab or PCR
cabinet. Filter tips are recommended for all pipetting steps.
1. Pulse-spin each tube in a centrifuge before opening.
This will ensure lyophilised primer and probe mix is in the base of the tube and is not
lost upon opening the tube.
2. Resuspend the primer/probe mix in the RNase/DNAse free water supplied,
according to the table below.
To ensure complete resuspension, vortex the tube thoroughly.
Component - Resuspend in water Volume
Internal Control primer/probe mix CY5 (BROWN) 165 µl
3. Resuspend internal control in 500 µl Template Preparation Buffer.
Component - Resuspend in template preparation buffer Volume
Internal Control RNA template (BLUE)* 500 µl
* This component contains high copy number template and is a VERY significant
contamination risk. It must be opened and handled in a separate laboratory environment,
away from the other components.
Detection of 20H/501Y.V2 (South Africa variant) 11
SNPsig kit handbook Path-SNPsig-SARS-CoV-2-20H/501Y.V2—STED version 1.03
Published Date: 24/03/20214. Prepare a complete genotyping reaction mix including the internal control primer/probe
mix and the resuspended internal control template according to the table below:
Include sufficient reactions for all samples, positive and negative controls.
Component Volume
Onestep lyophilised mastermix 10 µl
SARS-CoV-2 20I/501Y.V1 primer/probe mix (BROWN) 1 µl
Internal Control primer/probe mix (BROWN) 1 µl
Internal Control RNA template 1 µl
RNase/DNAse free water (WHITE) 2 µl
Final Volume 15 µl
5. Follow qPCR amplification directions on page 8 with the exception that fluorogenic
data should be collected through the FAM, HEX and Cy5 channels.
Detection of 20H/501Y.V2 (South Africa variant) 12
SNPsig kit handbook Path-SNPsig-SARS-CoV-2-20H/501Y.V2—STED version 1.03
Published Date: 24/03/2021References
1. World Health Organisation (WHO). Coronavirus disease (COVID-19) [Internet]. [cited 2021 Jan 13]. Available from:
https://www.who.int/emergencies/diseases/novel-coronavirus-2019
2. Hadfield J, Megill C, Bell SM, Huddleston J, Potter B, Callender C, et al. NextStrain: Real-time tracking of pathogen
evolution. Bioinformatics [Internet]. 2018 [cited 2021 Jan 13];34(23):4121–3. Available from:
https://pubmed.ncbi.nlm.nih.gov/29790939/
3. Tang X, Wu C, Li X, Song Y, Yao X, Wu X, et al. On the origin and continuing evolution of SARS-CoV-2 [Internet].
[cited 2021 Jan 13]. Available from: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-
reports/
4. Tegally H, Wilkinson E, Giovanetti M, Iranzadeh A, Fonseca V, Giandhari J, et al. Emergence and rapid spread of a
new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in
South Africa. medRxiv [Internet]. 2020; Available from: https://doi.org/10.1101/2020.12.21.20248640
5. Cele S, Gazy I, Jackson L, Hwa S-H, Tegally H, Lustig G, et al. Escape of SARS-CoV-2 501Y.V2 variants from
neutralization by convalescent plasma. medRxiv [Internet]. 2021; 2021.01.26.21250224. Available from:
https://www.medrxiv.org/content/10.1101/2021.01.26.21250224v1
6. Andreano E, Piccini G, Licastro D, Casalino L, Johnson N V, Paciello I, et al. SARS-CoV-2 escape in vitro from a
highly neutralizing COVID-19 convalescent plasma. bioRxiv [Internet]. 2020;2020.12.28.424451. Available from:
http://biorxiv.org/content/early/2020/12/28/2020.12.28.424451.abstract
7. Greaney AJ, Loes AN, Crawford KH, Starr TN, Malone KD, Chu HY, et al. Comprehensive mapping of mutations to
the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human serum antibodies. [cited 2021
Jan 19]; Available from: https://doi.org/10.1101/2020.12.31.425021
8. Wu K, Werner AP, Moliva JI, Koch M, Choi A, Stewart-Jones GBE, et al. mRNA-1273 vaccine induces neutralizing
antibodies against spike mutants from global SARS-CoV-2 variants. bioRxiv [Internet]. 2021 [cited 2021 Jan 29];
Available from: https://doi.org/10.1101/2021.01.25.427948
9. Novavax Inc. - IR Site. Novavax COVID-19 Vaccine Demonstrates 89.3% Efficacy in UK Phase 3 Trial [Internet]. [cited
2021 Feb 1]. Available from: https://ir.novavax.com/news-releases/news-release-details/novavax-covid-19-vaccine-
demonstrates-893-efficacy-uk-phase-3
10. Johnson & Johnson. Johnson & Johnson Announces Single-Shot Janssen COVID-19 Vaccine Candidate Met Primary
Endpoints in Interim Analysis of its Phase 3 ENSEMBLE Trial [Internet]. [cited 2021 Feb 1]. Available from:
https://www.jnj.com/johnson-johnson-announces-single-shot-janssen-covid-19-vaccine-candidate-met-primary-
endpoints-in-interim-analysis-of-its-phase-3-ensemble-trial
11. World Health Organization. COVID-19 Weekly Epidemiological Update 22. World Heal Organ [Internet]. [cited 2021
Feb 01]. 2021;(January):1–3. Available from: https://www.who.int/docs/default-source/coronaviruse/situation-
reports/weekly_epidemiological_update_22.pdf
Detection of 20H/501Y.V2 (South Africa variant) 13
SNPsig kit handbook Path-SNPsig-SARS-CoV-2-20H/501Y.V2—STED version 1.03
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